ABSTRACT How chromatin domains such as euchromatin and heterochromatin are established and maintained are poorly understood. At the heart of chromatin organization are the histone proteins, which are deposited and removed by chaperone proteins. One such chaperone is Spt6, which is evolutionarily conserved and promotes transcription elongation through its interaction with the RNA polymerase II (RNAPII) C-terminal domain (CTD). During transcription, Spt6 mediates nucleosome reassembly in the wake of transcribing RNAPII and promotes H3K36 methylation, which recruits histone deacetylase and chromatin remodeling activities that maintain chromatin integrity in the coding region of genes. Our studies have now identified Spt6 as a central component of a novel regulatory circuit that maintains CTD phosphorylation; this circuit is itself controlled through Spt6 protein stability. We have also found that Spt6 is up-regulated during S phase, where it likely contributes to histone gene transcription and/or deposition at this critical stage. Further, we have uncovered an unexpected role for Spt6 in maintaining centromere identity through the potential targeting of the Psh1 E3 ligase that removes ectopically deposited histone H3 variant Cse4. Despite these findings, we know little about how Spt6 performs its chromatin-dependent activities. Thus, this proposal aims to elucidate the fundamental mechanisms by which Spt6 contributes to the maintenance of chromatin structure in the coding and regulatory regions of the genome - thereby contributing to the biology of Spt6 and to the mechanisms by which chromatin domains are established and maintained. To accomplish this goal, we will address the following key questions: 1) How is Spt6 regulated through its interactions with RNAPII and by post-translational modifications that occur on this protein? 2) Why is Spt6 cell cycle regulated, and what role does it play in S phase? 3) Does Spt6 function outside of its canonical role in transcriptional regulation to regulate other aspects of chromatin biology, in particular centromere identity? Given the conservation of Spt6 across evolution, our studies will significantly impact our understanding of the mechanisms that underlie transcriptional elongation and the maintenance of chromatin integrity that govern genome integrity. These studies will also be relevant to our understanding of Spt6 in human development and disease.